P616: THE LOSS OF IΚBΕ INHIBITOR ACCELERATES DISEASE DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA
Authors: C. Lenzi, J. Bordini, A. Pseftogkas, A. Morabito, G. Tsiolas, F. E. Psomopoulos, A. Campanella, M. Frenquelli, P. Ghia
Journal: HemaSphere (2022)

Background: Chronic lymphocytic leukemia (CLL) is an indolent B cell malignancy in which B lymphocytes accumulate in peripheral blood (PB), bone marrow (BM) and spleen (SP), as a result of a complex intertwining between microenvironmental stimuli and genetic defects. Molecular studies revealed recurrent mutations in a large number of genes, each present with distinct frequency in different subgroups of patients. Among these genes, NFKBIE, a negative regulator of the NF-κB pathway, has been found mutated in a relevant proportion of patients with CLL, mainly those belonging to the clinically aggressive subset #1, suggesting that a reduced expression of NFKBIE may contribute to CLL aggressiveness through constitutive NF-κB activation independent of BcR signaling. Aims: We aimed at studying the effects of NFKBIE in the pathogenesis of CLL using functional inactivation of the gene both in vitro and in vivo. Methods: We engineered the MEC-1 CLL cell line using CRISPR/Cas9 technology to generate NFKBIE- knock-out (KO) cell lines. We analyzed the effect of NFKBIE ablation studying in vitro cell proliferation and migration, gene expression, BcR signalling pathway, and response to treatment with the BTK inhibitor Ibrutinib. We also evaluated the relevance of NFKBIE manipulation in vivo by injecting the engineered cells into RAG2-/- γc-/- immunodeficient mice. In addition, we generated NFKBIE-/wt mice and crossed them with Em-TCL1 mice, a validated CLL mouse model, to generate immunocompetent Em-TCL1-NFKBIE-/wt. We analyzed disease expansion by flow cytometry, measuring the amount of malignant B cells in the PB, SP and BM. Results: MEC-1 cells carrying NFKBIE ablation showed faster proliferation in vitro as compared to controls (p<0.05). RNAseq analyses indicated de-regulation of migratory pathway that was functionally confirmed by migration experiments with the specific chemokines (i.e. CCL19, CCL21 and SDF1). In particular, the percentage of migrated KO cells was significantly higher than that of control cells, either in the presence or the absence of each of the three chemokines. As predicted by the function of the gene, biochemical analyses in KO cells revealed the increase of cRel and p50 protein levels and the increase of p65 phosphorylation, all NF-κB transcription factors indicating unabated cellular activation. Interestingly, in vitro culture of the cells in the presence of Ibrutinib showed that NFKBIE KO cells are more resistant to apoptosis induction than control cells (p<0.05). The effect of NFKBIE manipulation was also more evident in vivo. Xenografts generated with MEC-1 NFKBIE KO cells in RAG2-/- γc-/- immunodeficient mice showed an increased disease expansion in the SP and BM (p<0.05) and a shorter survival (p<0.05) than mice injected with control cells. In immunocompetent Em-TCL1 mice, which develop the CLL-like disease with aging, preliminary analysis at 3, 6 and 9 months of age showed higher malignant cells expansion in PB of Em-TCL1-NFKBIE-/wt compared with control Em-TCL1 mice. Summary/Conclusion: The loss of NFKBIE function appears to be associated with a more aggressive disease in all CLL models tested, both in vitro and in vivo, accelerating disease development under CLL pro-tumorigenic conditions. In addition, it appears to be potentially involved in the onset of resistance to the BTK inhibitor Ibrutinib, thus providing an alternative mechanism that may explain drug failure in the resistant patients who do not carry BTK/PLCG2 mutations.